臺灣博碩士論文加值系統

蛋白質磷酸化在細胞訊息傳遞及功能調控上扮演重要關鍵角色。細胞再生物體中的增殖(proliferation)、分化(differentiation)或受到環境影響而產生的變化。為了更進一步了解生物體的話，發展磷酸化蛋白質的定性及定量的分析方法是必要的。在人類疾病中如帕金森氏症，也發現蛋白質磷酸化異常現象。
在此論文中，我們發展一套新的定量方法可同時分析磷酸化蛋白質相對量與絕對磷酸化程度。我們先將樣品經過酵素消化後利用金屬親合層析結合質譜技術進行磷酸化胜肽的鑑定。之後，將樣品分成兩個不同狀態(控制組及實驗組)，實驗組的磷酸化胜肽的Serine、Threonine以及 Tyrosine 胺基酸上做去磷酸化反應，利用氫氟酸將磷酸根從 Serine、Threonine以及 Tyrosine上移除。接著，在兩種不同狀態的胜肽分別接上isotope tag for relative and absolute quantification (iTRAQ)試劑。將兩樣品混合利用LC-MS/MS分析，我們在跟鑑定到的磷酸化胜肽資料庫比對，找出去磷酸化的胜肽利用iTRAQ進行定量分析。而磷酸化程度的計算，是計算同位素標定後的去磷酸化胜肽以及蛋白質變化量是計算相同蛋白質中非磷酸化胜肽的離子強度得到。
實驗結果得到在Jurkat T-cell細胞質中鑑定1435個磷酸化蛋白質中及3862個磷酸化胜肽且鑑定到15個磷酸化蛋白質的磷酸化程度，也在H460 cell中鑑定到10個磷酸化蛋白質的磷酸化程度。這結果證實此技術不止可鑑定出磷酸化的胜肽，更重要的是，可定量分析少量磷酸化胜肽的磷酸化程度，且這方法可同時針對蛋白質做定量分析，將來能廣泛應用在蛋白質體學中磷酸化蛋白質的大規模鑑定和定量分析。

Cellular processes such as proliferation, differentiation, and adaptation to environmental changes are regulated by protein phosphorylation. Qualitative and quantitative analysis of protein phosphorylation is important to elucidate the “molecular switch” in cellular processes. Increasing numbers of human diseases such as neurodegenerative diseases were discovered to involve mutations, overexpression, or malfunction of protein kinases and phosphatases as well as their regulators and effectors.
In this study, we developed a new quantitation strategy to determine the relative change and the absolute degree of protein phosphorylation. Here, we established a phosphoprotein database of Jurkat T-cell by utilizing immobilized affinity chromatography (IMAC) in combination with LC-MS/MS analysis. For quantitation analysis, the proof-of-concept experiment was performed on digested β-casein peptides mixtures with various protein concentrations and various degree of phosphorylation. One half of β-casein digested mixtures in which the phosphate group of phosphoseryl, phosphothreonyl and phosphotyrosyl residues was removed by HF treatment. HF treatment peptides and the other half of peptides were labeled with isotope tag relative and absolute quantification (iTRAQ) reagent, respectively. After iTRAQ labeling, those peptides mixtures were combined for analyzed by LC-MS/MS. The change in protein phosphorylation degree can be detected through the change in the intensity of dephosphopeptide and the change in protein level can be measured by in the intensity of non-phosphopeptide from the same protein.
In this study, the preliminary result identified 1435 phosphoproteins with 3862 phosphopeptides, about 55.6% of the identified peptides from Jurkat T-cell were found to be phosphorylated. We identified 12 phosphoproteins phosphorylation degree from Jurkat T-cell, and 10 phosphoproteins phosphorylation degree from H460 cell. The results illustrated capability of the IMAC-iTRAQ strategy to not only identify the phosphorylated site, but also more importantly to permit the quantitation for phosphorylation degree of low abundance phosphopeptides. This method is also capable of protein quantitation on a global basis. Overall, the results exemplify the application of the IMAC-iTRAQ strategy approach and demonstrate its potential utility for proteome-wide site-specific phosphoprotein identification and quantitation.

謝誌......................................................Ⅱ
中文摘要..................................................Ⅲ
ABSTRACT..................................................Ⅴ
CONTENTS..................................................Ⅶ
LIST OF FIGURES...........................................Ⅸ
LIST OF TABLES..........................................ⅩⅠ
ABBERVIATIONS...........................................ⅩⅡ
CHAPTER 1 INTRODUCTION.....................................1
1-1 Phosphoproteomics...................................1
1-1.1 Post Translational Modification...................1
1-1.2 Significance of Protein Phosphorylation...........2
1-1.3 Complexity and Analytical Challenges of Protein Phosphorylation ........................................3
1-2 Analysis of Protein Phosphorylation.................4
1-2.1 Qualitative Analysis of Protein Phosphorylation...4
1-2.1.1 Determination of Protein Phosphorylation Site by
Classical Methods................................4
1-2.1.2 Determination of Protein Phosphorylation by Mass Spectrometry...............................................5
1-2.2 Mass Spectrometry-Based Strategies for Quantitative Analysis of Protein Phosphorylation...........9
1-2.2.1 Tradiational Quantitation of Protein Phosphorylation............................................9
1-2.2.2 Absoulte Quantitation of Protein Phosphorylation...........................................11
1-2.3 Purpose of this Study............................12
CHAPTER 2 EXPERIMENTAL METHODS............................14
2-1 Materials..........................................14
2-2 Instrumentation....................................16
2-2.1 High-Performance Liquid Chromatography...........16
2-2.2 MALDI-TOF MS (Voyager)...........................17
2-2.3 Hybrid Quadrupole TOF (QSTAR)....................18
2-3 Preparation of Jurkat T Cell.......................19
2-3.1 Cell Culture and Preparation.....................19
2-3.2 Subcellular Fractionation........................20
2-4 Protein Quantitation...............................21
2-4.1 BCATM Protein Assay Kit..........................21
2-4.2 Coomassie (Bradford) Protein Assay Kit...........22
2-4.3 SDS-PAGE.........................................22
2-4.4 In-Gel Digestion.................................23
2-5 Desalting and Concentration........................24
2-5.1 Zip-Tip..........................................24
2-5.2 Macro-trap.......................................24
2-6 Peptides Dephosphorylation.........................25
2-7 iTRAQ Labeling Reaction............................25
2-8 Immobilized Metal Affinity Chromatography..........26
2-8.1 Preparation of IMAC Column.......................26
2-8.2 IMAC Purification of Phosphopeptides.............26
2-9 Strong Cation Exchange Column (SCX)................27
2-10 Capillary LC-MS Analysis and Data Analysis........29
2-10.1 Capillary LC-MS Analysis........................29
2-10.2 Protein Identification..........................29
CHAPTER 3 RESULTS.........................................30
3-1 The Overview of iTRAQ-IMAC Strategy................30
3-2 Optimization for the De-phosphorylation of Phosphopeptides...........................................31
3-2.1 Dephosphorylation Efficiency.....................31
3-2.2 Sample Recovery after HF Treatment...............32
3-3 Performance of iTRAQ-IMAC Strategy.................34
3-4 Quantitation for the Level of Phosphorylation with Different Protein Concentrations..........................35
3-5 Analysis of Protein Phosphorylation Degree from Jurkat T Cell.............................................38
3-5.1 Overview for the Analysis of Complex Protein Mixture from Cell Lysate..................................38
3-5.2 Profiling of Protein Phosphorylation Using IMAC-LS-MS........................................................40
3-5.3 Quantification of phosphorylation degree.........41
CHAPTER 4 Discussion......................................43
CHAPTER 5 COCLUSION AND PROSPECTS.........................47

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